A Self-Monitering of Blood Glucose using an electrochemical biosensor has been widely used. The biosensor utilizes enzymes whose substrate is glucose, such as glucose dehydrogenase (hereinafter abbreviated as “GDH”) and glucose oxidase (hereinafter abbreviated as “GO”). GO is highly specific to glucose and is excellent in thermostability. However, a measurement method of using GO is easily affected by dissolved oxygen in a measurement sample, and such a problem that the dissolved oxygen gives an adverse effect on a measurement result has been pointed out.
On the other hand, as an enzyme that does not receive an effect of dissolved oxygen and acts on glucose in absence of NAD (P), GDH having pyrroloquinoline quinone (PQQ) as a coenzyme (hereinafter abbreviated as “PQQ-GDH”) has been known (for example, see Patent Documents 1 to 3). However, PQQ-GDH has problems such that (1) PQQ is easily dissociated from an enzyme, (2) specificity of glucose is low, and (3) since it is generally present in a membrane fraction, extraction and isolation operations thereof are performed with difficulty.
As an enzyme that does not receive an effect of dissolved oxygen and acts on glucose in absence of NAD (P), other than PQQ-GDH, a glucose dehydrogenase having a flavin adenine dinucleotide as a coenzyme (hereinafter abbreviated as “FAD-GDH”) has been known. FAD-GDH has been obtained respectively from Aspergillus oryzae (Non-patent Documents 1 to 4, Patent Document 4) and Aspergillus terreus (Patent Document 5) so far. It has been known that, as general characteristics of FAD-GDH, reactivity to xylose is comparatively high (for example, in the case of FAD-GDH disclosed in Patent Document 5, reactivity to xylose is about 10% of action property to glucose) and an optimum temperature is high (for example, the optimum temperature of FAD-GDH disclosed in Patent Document 4 is about 60° C.). Note that enzyme modification has been vigorously tried for the purpose of enhancing practicality, and the like. Documents that report modification of FAD-GDH are shown below (Patent Documents 6 to 9).
Recently, a conformational analysis of FAD-GDH was carried out using the structure of GO, and it was reported that Glu414 and Arg502 are important for substrate recognition (Non-patent Documents 5 and 6).